Vaccinia uracil-DNA glycosylase (VUG) removes uracil from DNA. It is coded for in the genome of the vaccinia virus, and is essential for viral proliferation. The crystal structure of human uracil-DNA glycosylase (UDG) bound to DNA shows that the enzyme binds extrahelical uracil. DNA binding involves electrostatic orientation along the UDG active site, insertion of amino acid 272 via the DNA minor groove, cn of the DNA backbone flanking uracil, and specific recognition of phosphate, deoxyribose, and uracil flipped-out via the DNA major groove. VUG is homologous to the human enzyme, and solution of the structure by molecular replacement is feasible. However, the sequence differences between VUG and UDG are significant enough to change the nature of the active site as evidenced by the observation that uracil glycosylase inhibitor protein (UGI),which inhibits most UDGs including human, does not inhibit VUG.